Project Summary Many essential proteins are inserted into membranes or secreted. Because they are synthesized in the cytosol, these proteins must cross a lipid bilayer to reach their destination and become functional. The majority of proteins bound for secretion or membrane insertion transits through the universally conserved Sec translocon. The Sec pathway allows proteins destined for export from the cytosol to cross the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in bacteria. The essential role of Sec-dependent translocation in many cellular pathways makes elucidation of the underlying molecular mechanisms an important task. Substrates of the Sec system include virulence factors and antibiotic-inactivating enzymes in bacteria, and range from insulin to antibodies in animals. Perturbations in the Sec-pathway can lead to numerous diseases, including cancer and diabetes. A mechanistic understanding of the translocation process can fuel the development of drugs that target this central cellular pathway. Sec-dependent protein translocation has been studied extensively with biochemical and structural approaches, characterizing many of its components. However, the dynamics of the process are not well understood. During translocation, the translocon channel must allow the polypeptide to move while maintaining a permeability barrier for ions and other solutes. How the channel interacts with polypeptide substrates to achieve these seemingly conflicting requirements is not known. Another key question that has remained unanswered is how the molecular machines that associate with the translocon convert chemical energy into the mechanical work that powers translocation. Many of the important outstanding questions concerning the Sec translocation system could be answered if it were possible to follow the passage of a protein through the channel in real-time with high spatial and temporal resolution, but this capability is not presently available. Single-molecule approaches, enabling observation and manipulation of individual macromolecules, have provided unprecedented insights into biological mechanisms. I propose to investigate the mechanisms of Sec- dependent protein translocation with optical tweezers. This approach enables us to unravel the mechanisms underlying Sec-dependent protein translocation. Our studies will yield new and exciting insights into the mechanisms employed by the translocation machinery to transport proteins out of the cytosol.